The human airway epithelium (HAE) represents the entry port of many human respiratory viruses, such as human coronaviruses (HCoVs). Nowadays, four HCoVs, HCoV-229E, HCoV-OC43, HCoV-HKU1 and HCoV-NL63, are known to circulating worldwide, which cause upper and lower respiratory tract infections in non- and hospitalized children. Still studies on fundamental aspects of these HCoV infections at the primary entry port, such as cell tropism are seriously hampered by the lack of a universal culture system, or suitable animal models. To expand the knowledge on fundamental virus-host interactions of all four HCoVs at the site of primary infection, we used pseudo-stratified HAE cell cultures to isolate and characterize clinical representative HCoV strains directly from nasopharyngeal material. Ten contemporary isolates were obtained representing HCoV-229E (n = 1), HCoV-NL63 (n = 1), HCoV-HKU1 (n = 4) and HCoV-OC43 (n = 4). From each strain we analyzed the replication kinetics and progeny virus release on HAE cell cultures derived from different donors. Surprisingly, by visualizing HCoV infection using confocal microscopy, we observed that HCoV-229E employs a target cell tropism for non-ciliated cells whereas HCoV-OC43, HCoV-HKU1 and HCoV-NL63 all infect ciliated cells. Collectively, we demonstrate that HAE cell cultures, that morphologically and functionally resemble human airways in vivo, represent a robust universal culture system to isolate and compare all contemporary HCoV strains.